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"... Abstract. After DNA viruses enter the nucleus, they initiate a transcriptional cascade which is followed by replication. We investigated whether these processes take place at specific nuclear sites or, as suggested by the mode of entry, randomly throughout the nucleus. Three distinct nuclear domains ..."

Abstract. After DNA viruses enter the nucleus, they initiate a transcriptional cascade which is followed by replication. We investigated whether these processes take place at specific nuclear sites or, as suggested by the mode of entry, randomly throughout the nucleus. Three distinct nuclear domains, nuclear factor-1 sites, coiled bodies, and nuclear domain 10 (ND10), were used as markers to investigate the relative position of DNA virus replication sites. We found that all three nuclear domains had a very high spatial correlation with each other in uninfected cells. After adenoviral infection, nuclear factor 1 and coiled bodies were found associated with some viral replication domains. Simian virus 40 begins replication adjacent to ND10 but adenovirus 5 and herpes simplex type 1 modified ND10s

...with deletions were propagated on the Veto-derived complementing cell line W162 (Weinberg and Ketner, 1983). The wild-type Ad5, H5 7001, H5 1101, H5 338, and Ad CB were propagated in human 293 cells (=-=Graham et al., 1977-=-). The E4 ORF3 expression vector was developed from a PCR-derived fragment by inserting it into a plasmid containing the cytomegalovirus promoter. Cells were transiently transfected by Ca-phosphate pr...

"... The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated vira ..."

The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in trans-duction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols. Adeno-associated virus (AAV) is a nonenveloped virus with

O.Gires and F.Kohlhuber contributed equally to this work Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein–Barr virus. Molecular and biochemical approaches demon-strated that LMP1 usurps cellular signaling pathways resulting in the induction of NF-κB and AP-1 via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1’s C-terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphoryla-tion of JAK3 within minutes after crosslinking of a conditional NGF-R:LMP1 chimera and is a pre-requisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

"... Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipul ..."

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAs were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells. Adenoviruses are generally associated with benign patholo-gies in humans and are characterized by a number of features which make them particularly attractive as gene transfer vec-tors for gene therapy or immunization purposes (7, 15, 21, 35). Most vectors are based on the adenovirus type 5 (Ad5) back-bone in which an expression cassette containing the foreign

... region 1 (E1) or early region 3 (E3). Viruses in which E1 has been deleted are defective for replication and are propagated in the human 293 complementation cells, providing the E1 products in trans =-=(16)-=-. Methods to construct such recombinant adenoviruses are well documented (3, 14). They usually consist of a first step of subcloning of the exogenous expression cassette into a segment of the viral ge...

...ance of the virion DNA (15, 18). The biological assay most widely used for quantification of E1-deleted adenovirus vectors is the plaque assay on 293 cells, which support replication of these vectors =-=(8, 9)-=-. Detection of the virion in this assay involves infection of a cell in a cultured monolayer by a virion suspended in the medium above the cells followed by virion replication and spread of infection ...

"... Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simp ..."

Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named �5. We engineered a cis-acting selection into �5 by flanking its packaging site with loxP sites. When �5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of �5 was reduced to 5 % of the wild-type level. To make a new E1-substituted virus, we used �5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows �5 in the presence of Cre recombinase. To make a gutless virus, we used �5 asa helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between �5 and the gutless viruses within their packaging domains.

"... Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV- ..."

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1.

...n PBMC. Here we used acute/early infection Env clones in pseudoviruses that were produced by transfection in 293T cells and assayed in JC53-BL cells; both of these cell lines are epithelial in origin =-=(40, 45)-=-. These new technologies afford significant technical advantages for assay standardization, validation, and high throughput, making them an attractive alternative to PBMC assays. However, the type of ...